AVIAN influenza, or bird flu, and severe acute respiratory syndrome (SARS) were recent demonstrations of how the human-animal interface could result in the emergence of new infections or unknown forms of old diseases zoonosis as it is termed that could assume pandemic proportions. The ongoing swine flu outbreak, which at present is in pandemic alert phase 5 (one notch below a true pandemic as per the World Health Organisations classification), is a classic example of how this zoonotic bridge is, in fact, a two-lane highway.
There are three genera of influenza viruses that cause human flu: A, B and C, of which C is rare. Swine flu is predominantly of type A, and type B has not been reported in pigs. Influenza A virus strains are categorised according to two surface proteins of the virus: haemagglutinin (H) and neuraminidase (N). All influenza A viruses contain both H and N, with strains assigned an H number and an N number depending upon the forms of the proteins. There are 16 H and nine N known subtypes, but only H1N1, H1N2 and H3N2 are currently in general circulation among humans. The strain responsible for the 1918 Spanish flu pandemic, which infected nearly one-quarter of the worlds population and caused 20 million to 50 million deaths, belonged to the A(H1N1) subtype. This subtype has now become the most common cause for the common flu in humans. In fact, some strains of A(H1N1), including the 1918 pandemic strain, have become endemic in humans. Less virulent strains are responsible for a large fraction of all seasonal influenzas. Other subtypes are mostly found in other species.
The viral genome, however, is unstable and, therefore, highly susceptible to rapid mutation and reassortment of the eight genetic segments of which it is composed. Hence, the structure of these proteins differs from strain to strain. The current swine flu virus has been decoded to be a new strain of the influenza virus subtype A(H1N1), and the various isolates from different parts of the world have been found to be genetically similar. Though different strains of A(H1N1) are the causative viruses for human flu and swine flu, how, where and when the new strain, which threatens to cause a pandemic, originated remains a mystery.
Pigs seem to be unusual in that they can be infected at the same time with influenza strains associated with three different species: pigs, birds and humans. This makes pigs a potential mixing vessel where influenza genes might swap genes (genetic reassortment) to produce new reassortant virus strains to which humans may have no immunity. Genetic reassortment occurs when different strains of influenza virus infect a single cell. The virus that has been implicated in the current wave of swine flu infection is one in which four distinct genetic changes, or antigenic shifts, have occurred over the years to emerge as a totally new strain. The strain is seen to be made up of a unique combination of gene segments of human, avian and swine influenza virus strains that had not been previously seen in swine or human influenza virus strains, and therefore, no preventive vaccine against it exists at the moment.
Though it was only in 1931 that the influenza virus was identified as the cause of febrile respiratory illness in pigs, it was actually during the 1918 flu pandemic that pigs got infected with A(H1N1). It is believed that humans infected pigs during the pandemic reverse zoonosis because swine influenza was noted as a disease amongst pigs only after the first large outbreak in humans. Subsequently, the human and the swine A(H1N1) viruses have evolved into divergent A(H1N1) viruses. In fact, a recent editorial in The New England Journal of Medicine (NEJM) said, The current situation is not 1918 again, it is 1918 continued in that we are still being infected with remnants of the 1918 pandemic influenza virus.
Between the 1930s and the 1990s, this classic swine flu virus A(H1N1) underwent little change. The virus is endemic in the United States, and outbreaks have been reported in North America, South America, Europe (including the United Kingdom), Kenya and parts of East Asia. Today, swine flu is known to be caused by A(H1N1), A(H1N2), A(H3N1), A(H3N2) and A(H2N3), but H1N1, H1N2 and H3N2 are the most common worldwide. In addition, however, since 1998, multiple strains and subtypes of triple reassortant A(H1) swine flu viruses which include combinations of North American avian, human A(H1N1) and A(H3N2), and classic swine influenza virus gene segments have emerged and become predominant among North American pig herds.
The fact that humans are being infected with swine flu is in itself not new. Symptoms of infection with classic swine flu in humans are often indistinguishable from those of human flu virus infection. According to a recent paper by Vivek Shinde and others in the NEJM, cases and clusters of human infections with swine flu viruses have been reported sporadically since the 1970s. Worldwide, in the past 35 years, there have been more than 50 documented cases, including seven deaths, of swine flu virus in humans, most caused by the classic A(H1N1). And until April 2009, only limited and unsustainable human-to-human transmission have been reported.
There were two instances of human infection with the triple reassortant swine flu viruses A(H1) in Canada in 2005. In December 2005, the first human infection with the triple reassortant A(H1) virus was found in the U.S. According to Shinde and associates, between December 2005 and February 2009, there were 12 notified cases of human infection with the triple reassortment virus, including one as recently as January 2009 whose final determination is, however, pending. Of these, nine had exposure to pigs and one was suspected to be a case of human-to-human transmission. All the patients had illness of the lower respiratory tract and unusual influenza signs such as diarrhoea in some of them, including those who had been healthy. All the 11, however, recovered from the illness.
Significantly, some of these symptoms, particularly the respiratory distress, have been seen in the current outbreak as well.
Although uncommon, says the paper, such cases are likely to continue to occur sporadically, since triple reassortant swine flu A(H1) viruses are endemic in North American swine herds. Given the recent events, they add, [T]he generation of novel influenza viruses with other human and influenza viruses may be inevitable. In this context, the possibility of novel influenza viruses causing epidemic and pandemic disease in large populations of immunologically susceptible humans remains a major ongoing public threat. While this remark may seem to be in hindsight, what is clear is that this is not the last of such new swine flu viruses that one will see.
The current outbreak differs in important ways from earlier human infections due to existing swine flu strains, classic and triple reassortant. Firstly, this new influenza strain has never been reported in pigs earlier. It seems to have emerged in humans, and given the rapidity of its spread within communities as well as between geographically separated populations, it seems to be able to sustain itself through human-to-human transmission. There has also been an interesting episode of the new strain being passed back to the pigs reverse zoonosis by an infected farm worker in Alberta, Canada, thus completing the zoonotic cycle.
The case histories of the earliest patients suggest that the first case of this new swine flu probably emerged in Mexico though it was first detected in California, U.S., with many cases in other countries, probably associated with travels from these countries. The new strain identified in the U.S. was later found to be genetically similar to the virus subsequently isolated from Mexican patients. There is, however, no firm epidemiological evidence at this point to say that the virus first emerged in Mexico.
As of May 13, according to the WHO, 33 countries had officially reported 5,728 cases of the new infection in humans. Of these, Mexico reported 2,059 laboratory-confirmed cases, with 56 deaths. The U.S. reported 3,009 confirmed cases, with three deaths. Canada reported 358 confirmed cases, including one death. Costa Rica reported eight confirmed cases, including one death. The other 29 countries reported no deaths. As of now, no case has been reported from India. According to the Ministry of Health and Family Welfare, screening of passengers coming from affected countries is being done at the 21 international airports in the country, and 4.2 lakh passengers have been screened as of May 12. A total of 40 suspected cases were tested but were found to be negative for the virus. Samples from two more cases are under test, and three persons have been referred to the identified medical facility.
The first U.S. detection of the virus was from two cases of febrile respiratory illness in children a boy aged 10 and a girl aged nine who live in adjacent counties in southern California. The Centres for Disease Control and Prevention (CDC) determined the genetic structure of their virus isolates on April 14 and 17 respectively and found that they were because of a A(H1N1) swine flu virus. The viruses from the two cases were found to be genetically similar but substantially different from the A(H1N1) strains that had been seen earlier. The children, who developed influenza-like symptoms in March-end, had no contact with pigs. The family members of both too also subsequently developed influenza-like symptoms, such as fever, cough and headache, which, however, did not sustain. These were probably the first indications that human-to-human transmission had occurred with the new swine flu virus. The virus strain has been termed by the CDC as A(H1N1) swine-origin influenza virus (S-OIV).
In March and early April, which was the end of the seasonal influenza period, Mexico experienced outbreaks of respiratory illness, including pneumonia, and increased reports started coming from several areas of the country of patients with influenza-like illness (ILI). Nothing untoward was suspected as it only seemed to be a rather unusual case of a prolonged period of seasonal influenza, the last outbreak of which was in La Gloria, Veracruz State, in the last two weeks of March. But the pneumonia cases were still a concern, according to Celia Alpuche Aranda, the head of Instituto de Diagnostico y Referencia Epidemiologicos (InDRE) in Mexico City. According to her, this is the only laboratory in Mexico with the capability to test for influenza subtypes, whereas the other public health laboratories can only tell whether the virus type is A or B.
An interesting thing about the Veracruz outbreak was that symptoms began to show only after four or five days, an incubation period that is longer than that for the usual seasonal flu, whereas the sensitivity of the immunofluorescence test on the nasopharyngeal swabs that they used was low after 72 hours. So most of the samples were negative, which led to the InDRE receiving several samples from patients from La Gloria who started showing symptoms in early April. The exact subtype of a subset of these samples could, however, not be identified because of the limitations of the technique being used, which were basically primed for known subtypes only caused by the ongoing seasonal flu.
These untypable samples were then sent on April 21 to the CDC and to the Public Health Agency of Canada (PHAC) for assistance in subtyping. It was on April 23 that Mexico learnt that an epidemic of a new swine flu virus, A(H1N1) S-OIV, was circulating in the country, which was the same as what had been found in the U.S. But, sensing that something was unusual about the Veracruz outbreak of ILI, Mexico had notified the Pan American Health Organisation (PAHO) of the WHO on April 12 itself. On April 17, triggered by the isolated case of a 37-year-old woman who developed ILI, including atypical pneumonia, on April 4, Mexico intensified its influenza surveillance mechanism, which resulted in a rapid increase in the number of suspected cases, along with hospitalisation for severe acute respiratory illness. Both the CDC and Canada later helped Mexico set up real-time reverse-transcription polymerase chain reaction (RT-PCR) equipment in various laboratories to test for swine flu directly.
The earliest sample from La Gloria was from a boy who began to develop symptoms on April 1, and the case has now been labelled the index case. This case has led to a lot of Mexican and international press reports about ground zero being the largest pig farm in Mexico, Granjas Carroll de Mexico in Perote (which is near La Gloria), a subsidiary of the U.S. multinational pork giant Smithfield Foods. The suspicion gained ground because the farm already has a poor reputation for overcrowding of pigs, poor hygiene and its intensive farming practices. In fact, a faeces lagoon in the farm was being identified as source of the infection. However, the company dismissed the allegation, and the Mexican agricultural authorities, too, apparently did not detect any problems in the farm, such as infected employees or infected pigs, according to Celia Alpuche Aranda.
The more likely scenario, according to her, is that of migration into Mexico of an infected person. This belief arises from the fact that the other earliest case was of an Oaxaca woman who subsequently died. Oaxaca and Perote as well as San Luis Potosi, where there have been a lot of cases during the active swine flu epidemic period, are far apart and are well known for migration, according to Celia Alpuche Aranda. Its hard to believe that its going to be associated with this farm[and] the authorities have thoroughly investigated it, Celia Alpuche Aranda told ScienceInsider, a blog site of Science magazine. If this is the true scenario, it would be extremely difficult to pin down the origin.
But does the genetic make-up of the new virus A(H1N1) strain offer any insights? The strain is actually the result of a second level triple reassortment. According to the members of the team in the U.S. investigating the nature of this new strain, the virus isolated on April 15 from the first U.S. case, the 10-year-old boy from California, was found to contain [six] genes [PB2, PB1, PA, HA, NP and NS] from triple reassortant swine influenza viruses that were known to circulate among swine herds in North America and two genes encoding the neuraminidase and matrix proteins [NA and M] that were most closely related to genes of viruses obtained from ill pigs in Eurasia.
According to CDC virologist Ruben Donis, under whom the genetic analysis was done, the new strain is almost equidistant from the U.S. and the Eurasian swine viruses. As mentioned earlier, the triple reassortant swine flu viruses were the reassorted ones in 1998 from the classic swine flu A(H1N1), human flu and North American avian flu viruses. In that sense, even though the virus is not found in pigs, the new virus strain is entirely of swine origin and the CDCs naming it A(H1N1) S-OIV and the popular term swine flu seem quite appropriate, notwithstanding the controversy about the name caused by protests from the pork industry as well as the unwarranted culling of pigs in Egypt.
An interesting question is how does Europe get to figure in this mixing? The genetic lineages of Asia and Europe mix quite a bit, said Donis, adding that European pigs need not come to North America; it is sufficient if infected people do. This again suggests that Mexico is not the origin of the strain. The amazing thing, said Donis, is the haemagglutinins we are seeing in this strain are a lonely branch that has been evolving somewhere and we dont know about it.
At present, 27 of the 31 Mexican states and the Federal District have now reported confirmed cases. Though the longer incubation period (of three to seven days), the clinical symptoms and the age distribution of cases (predominantly less than 18 years of age unlike seasonal flu) seem to be similar to those in the U.S. and Canada, the naive case fatality rate (CFR) seems to differ significantly. While it is slightly over 0.1 per cent in the U.S. and 0.3 per cent in Canada, it is much higher in Mexico. According to the WHO, outside Mexico, the new virus has caused very mild illness in otherwise healthy people, and nearly all cases of illness, and all the deaths, have been detected in people with underlying chronic conditions.
Why the outbreak in Mexico is somewhat different is not fully understood yet. Frank Plummer, the scientific director general of the National Microbiology Laboratory of the PHAC, which has been assisting the InDRE with the genetic analyses of Mexican samples and did the first full genetic sequencing of A(H1N1) S-OIV, has ruled out this to be the result of mutation. Though late reporting of the disease and delayed treatment could be responsible, the genetic make-up of the Mexican population could also play a role, according to him.
A recent report in Science by Christophe Fraser and associates, including Celia Alpuche Aranda and others from Mexico, however, cautions against a simplistic estimate of the CFR based on the data of suspected and confirmed cases as well as on suspected and confirmed deaths. The interpretation of these statistics, the authors point out, depends on the total number of infections, including those with mild infections or who are asymptomatic, which is currently unknown, given the absence of a specific serological test for the new H1N1 influenza strain and associated population level screening. The reported data may be an underestimate as surveillance had understandably focussed on severe cases, they point out. By analysing the outbreak in Mexico and early data on the international spread of the disease, they estimate that about 23,000 people were infected in Mexico by late April. This yields a CFR of 0.4 per cent, which is closer to the CFR in the U.S. and Canada, assuming that such screening biases were not so significant there.
Given the evidence in the U.S. and Mexico that the virus was capable of sustained human-to-human transmission and of spreading within communities, on April 29, the WHO raised the pandemic alert to phase 5. This is in contrast to bird flu, which peaked in 2006 and where the transmission was due almost entirely to direct contact between humans and birds. Phases 5 and 6 represent periods of time when the virus is beginning to spread from country to country and is becoming established. Phase 5 is when human-to-human transmission of the virus, from the community-level outbreaks of phase 4, spreads to at least two countries in one WHO region from where it is exported as isolated cases to other regions. Phase 5 is a strong signal that a pandemic is imminent and that the time to finalise the organisation, communication and implementation of mitigation measures is short. Phase 6 is characterised by community-level outbreaks in at least one other country in a different WHO region in addition to the criteria for phase 5. This phase is indicative that a pandemic is under way.
The chances of a pandemic would, of course, depend on the potency of the virus and its observed virulence and transmissibility. According to the WHO, the virus appears to be more contagious than seasonal flu. The secondary attack in the former are 22-33 per cent as compared with 5-15 per cent in the case of seasonal flu. An indication of the virulence comes from the molecular make-up of the virus and comparing it with the genetic markers for severity that have been identified from past outbreaks. Jonathan Allen and Tom Slezek of the Lawrence Livermore National Laboratory (LLNL), U.S., have studied the genetic sequences of the new virus and found that only about half of the 34 previously identified markers are present. This lack of similarity does not necessarily mean that the current H1N1 virus is not going to be a major problem, but it does suggest that it lacks many of the attributes that have made previous outbreaks deadly, Slezek said.
Though the outbreak surfaced only in April, researchers in the U.K. believe that the virus has been circulating for some time without causing any outbreak. This is indicative of the potency of the virus. Nicholas Grassly of Imperial College and Andrew Rambaut of the University of Edinburgh have analysed the rate of spread of the virus on the basis of the small mutations that have accumulated in almost two dozen genetic sequences of the new virus available up to now from the Mexican and American samples. If the rate at which genes mutate is about the same as for other H1N1 viruses, what one sees suggests that the virus has been around since January 2009 or even September 2008, though the limited sampling does imply considerable uncertainty.
The fact that the illness caused by the new strain is relatively mild, except in patients with chronic ailments, suggests it is a weak virus. Further, a model analysis how well the virus can spread from person to person an indicator of how many cases there may have been at a given time shows that the virus is not as transmissible as earlier pandemic strains but is more transmissible than seasonal flu. The model calculates a variable called the basic reproductive rate, or the R0, that is, the number of additional infections that an individual can cause. The infection will die out if the R0 is less than one. Fraser and others in their Science paper estimate the R0 to be 1.4 to 1.6 on the basis of epidemiological data. To explain the size of the current epidemic, in Mexico for example, this implies 14 to 73 generations of human-to-human transmission, according to the researchers.
The above may be some indication that a pandemic is perhaps not imminent. But history should warn us against such optimism and complacency. The 1918 flu pandemic started out mildly in spring and summer, when the R0 was estimated to be 1.45, only to return as a more lethal second wave in autumn with an R0 of 3.45. Whether one is likely to see a second wave depends on how well the new strain adapts to humans by the end of the year.
Another question is whether the new strain will mix with seasonal flu viruses to produce new, unpredictable strains. Particularly worrisome is the possibility of its mixing with H5N1, the avian flu virus that has been circulating since 2003 in Asia, and mutating into a form that spreads easily. Do not drop the ball in monitoring H5N1, WHO Director-General Dr. Margaret Chan told health officials on May 8. We have no idea how H5N1 will behave under the pressure of a pandemic.